This invention relates to capillary electrophoresis systems and particularly to such systems using analyte velocity modulation for improving detector sensitivity.
Capillary electrophoresis is a rapidly emerging high resolution separation technique, with broad applications in analytical chemistry, biochemistry adn molecular biology. It provides better resolution than liquid chromatography or one-dimensional gel electrophoresis. The capillary format is simpler to use than traditional slab gel elecrophoresis. Because it eliminates the delicate mechanical pump systems of liquid chromatography, an electrophoresis system is easier to maintain than liquid chromatographic instruments.
In free zone electrophoresis, the ionic analyte migrates in an electric field through a buffer-filled capillary. Separation is by ion mobility. The technique employs capillaries of 0.02-0.08 mm internal diameter. The capillaries are usually 500-1000 mm long. The operating voltage is typically 10,000-30,000 volts/meter. The driving voltage is provided by a hig voltage DC power supply. The analyte ions migrate along the capillary at slightly different rates and therefore reach the fixed detector at different times.
In a variant on the technique, the buffer contains micelles, which migrate through the capillary. Neutral analytes can partition between the moving micelles and the aqueous buffer. The neutrals, which all move by electro-osmosis at the same rate, are separated on the basis of their partition coefficients.
Gel-filled capillaries can be used to separate nucleic acids and denatured proteins. The gel filled capillaries are 0.2-0.3 mm diameter, and are 100-500 mm long. As with free zone electrophoresis, the operating voltages are 10,000-30,000 volts/meter. Typically, the gels and buffer systems of slab gel electrophoresis are used.
Capillary electrophoresis achieves hig resolution by use of high voltages and rapid migration rates. Ideally, the dominant zone-broadening (resolution limiting) effect is analyte diffusion. In practice, analyte adsorption on the capillary walls may also contribute. Much capillary electrophoresis research is devoted to techniques for eliminating or minimizing adsorption. At the usual migration times of 3-20 minutes, the zones migrate past the detector over a period of 2-10 seconds.
The capillaries must be narrow so that heat conduction is efficient and operating temperatures remain approximately constant. Rapid heat dissipation prevents convection, which would otherwise cause excessive zone broadening and loss of resolution. In free solution, it is known that convective effects are negligible if the capillary internal diameter is less than about 0.08 mm. The anti-convective effects of gels allows use of capillaries as wide as 0.3 mm internal diameter.
Absorbance detectors are the dominant detectors in capillary electrophoresis. They are used in many research applications and are employed in the one presently available commercial unit and in most or all of the commercial units currently under development. However, absorbance detectors are not as satisfactory as they have been in liquid chromatography. The narrow internal diameters of capillaries and small sample volumes employed are the source of insensitivity of absorbance detectors. The path length is short and the light throughput is small, so that such detectors are inadequately sensitive for many applications.
Fluorescence detectors are also known. Xenon flash lamps, arc lamps, mercury and zinc discharge lamps and He-Cd lasers have been used as excitation sources. Fluorescence is more sensitive than absorbance. However, most compounds of interest do not fluoresce, so a fluorescent derivative must be prepared before the separation or a post-capillary flow system must be used to prepare fluorescent derivatives of molecules as they exit the capillary. In either case, the basic simplicity of the capillary experiment is compromised.
Refractive index detectors have been described in the research literature. These devices are based on laser beam deflection. A laser beam, usually a He-Ne laser, is focused into the capillary. As the analyte passes through the beam, it causes the beam exiting the capillary to be deflected at a slightly different angle from the exit direction than when buffer only is present. These types of detectors are nearly universal in their application and can be implemented with a compact, inexpensive, low power laser, and an inexpensive beam position sensor. Such systems are especially applicable to protein and nucleic acid detection.
In another type of refractive index gradient detector, an extended laser beam or beam from a light emitting diode probes a length of capillary about 0.05-0.1 mm long. As the analyte zone passes through this region, its concentration gradient generates a refractive index gradient. This gradient causes the capillary to function as a prism of sorts. The exit angle of the beam is proportional to the refractive index or concentration gradient. This system probes a refractive index spatial gradient only. Accordingly, it does not suffer from some of the background drift problems of the conventional deflection detector, although drift due to beam pointing instability is still present.
Measurement of the change is conductance (inverse of electrical resistance) of the solution as the analyte passes by a pair of electrodes has been described. This technique is applicable to any ionic analyte whose conductance differs from that of the buffer. While the technique is nearly universal, it is difficult to implement because of the very small size of electrodes required.
Measurement of oxidation current with microelectrodes has also been reported. The technique gives very low detection limits, but the apparatus is delicate and difficult to fabricate. Mass spectrometry and Ruman spectrometry have been reported. These are special purpose and expensive techniques, which are indicated only when identification, rather than quantification is required.
Each of the detector systems described above are limited by fluctuations in the background signal from the buffer. The buffer is present at high concentration, typically about 0.01 M. The analyte may be present at concnentrations of 0.0001 M or less. The lower limit is whatever can be measured. The most important cause of background fluctuations is small (less than 1 degree Centrigrade) fluctuations in the temperature of the capillary. The temperature dependence of the measured phenomenon results in a large drift in the background signal.
A secondary cause of background fluctuations is movement of the capillary, caused by changes in the electrical double layer at the surfaces of the capillary. The capillary adjusts its position in an attempt to maximize the separation between adjacent cationic and anionic charges on the surface. This movement slightly changes the efficiency of signal excitation and/or collection and changes the background.
Finally, drifts in the signal excitation source intensity or position or the detector response also cause changes in the background. In laser deflection detectors, laser beam pointing stability and beam intensity stability are both contributors.
Most of these sources of background fluctuation occur at relatively low frequencies, 0.001-10 Hz. Their effects can be eliminated by restricting the measurement to substantially higher frequencies. Fluctuations in excitation source intensity or detector response may occur at any frequency. They can be minimized by measurement in a narrow band of frequencies. These considerations show that a system in which the desired signal is modulated at a frequency above 10 Hz and demodulated in a narrow band system, such as by using a synchronous demodulator (lock-in amplifier) would yield a detector in which the effects of background drift are much smaller than in conventional systems. Moreover, many forms of modulation will generate a derivative response. A derivative system will prove especially advantageous in capillary electrophoresis. In this high resolution technique, bands are narrow and dC/dx is correspondingly large.